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J Thromb Haemost. 2006 Jan;4(1):236-46.

CHO cells expressing the high affinity alpha(IIb)beta3 T562N integrin demonstrate enhanced adhesion under shear.

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1
INSERM U143, Le Kremlin-Bicetre, France.

Abstract

BACKGROUND:

Alpha(IIb)beta3-mediated platelet adhesive interactions in the vasculature, which are dependent on the functional state of this receptor, may be sensitive to shear forces.

OBJECTIVES:

To evaluate the influence of the alpha(IIb)beta3 affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity alpha(IIb)beta3 wild-type (wt) receptor to those expressing the high affinity alpha(IIb)beta3 T562N receptor.

MATERIALS AND METHODS:

We designed a real-time videomicroscopy adhesion assay for von Willebrand factor (VWF) or fibrinogen under flow conditions.

RESULTS:

At 50 s(-1), alpha(IIb)beta3 T562N supported higher cell adhesion to fibrinogen (63.3 +/- 2.9 cells/field) than alpha(IIb)beta3 wt (38.7 +/- 2.4 cells/field, P < 0.0001). At 100 s(-1), alpha(IIb)beta3 T562N mediated cell adhesion (40.5 +/- 3.8 cells/field), while alpha(IIb)beta3 wt did not (5.3 +/- 1.4 cells/field, P < 0.001), allowing to discriminate the efficiency of each receptor. Similar findings were observed for adhesion to VWF. Complete inhibition of cell adhesion to fibrinogen was achieved with 800 microM fibrinogen gamma-chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 microM) induced a dose-dependent cell detachment. These results suggest that the H12 motif allows initial attachment, in contrast to the RGDS site, which strengthens the stability of adhesion. Interestingly, compared with wt, a 10-fold lower concentration of RGDS was required to reach a similar reduction of cell adhesion mediated by alpha(IIb)beta3 T562N.

CONCLUSIONS:

Our data show that alpha(IIb)beta3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.

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